Receptor tyrosine kinases (RTKs) are the targets of numerous therapeutic agents used in cancer treatment. In particular, small molecule tyrosine kinase inhibitors (TKIs) that inhibit the intracellular catalytic activity of RTKs have had significant success in the clinic. Examples include erlotinib and gefitinib (which inhibit the EGF receptor), imatinib (which inhibits Kit), and sunitinib (which inhibits several RTKs). Many of the 58 RTKs in the human proteome have now been implicated in cancer and other diseases - either as oncogenic drivers or in mechanisms of resistance to existing targeted therapies. Accordingly, efforts are underway to extend approaches that have been successful for EGFR and Kit, for example, to other RTKs. This proposal focuses on a subset of RTKs that have been neglected as potential TKI targets because sequence analysis suggests that they contain inactive kinase domains. The intracellular domains of six RTKs (ErbB3, CCK4/PTK7, Ryk, EphB6, EphA10 and SuRTK106/STYK1/NOK) contain so-called 'pseudokinase' domains. These resemble normal tyrosine kinase domains but have sequence alterations in key motifs that are thought to render them catalytically inactive. In addition, two RTKs (Ror1 and Ror2) have unusual sequences in several of their catalytic motifs. Collectively, we term these the 'RTK pseudokinases'. 10% of the 518 protein kinases in the human proteome fall into this category. It has been proposed that RTK pseudokinases activate signaling pathways through regulated allosteric changes and/or scaffolding functions that control assembly of signaling molecules. Several biochemical studies have reported an absence of kinase activity. However, when assembled on membrane surfaces to mimic their oligomerization in an activated RTK, we have recently shown that the kinase domains of several RTK pseudokinases do have significant catalytic activity. In this proposal, we plan to test the hypothesis that the RTK pseudokinases are simply unusual kinases, but still use their catalytic activity for transmembrane signaling. Our approaches combine structural and enzymological analyses with studies of cell signaling and the effects of kinase inhibitors. Our Specific Aims are: 1. to test the hypothesis that pseudokinase domains in human RTKs have kinase activity, to determine its extent, its structural basis, and susceptibility to inhibition by ATP-competitive inhibitors. 2. To test the hypothesis that kinase activity of RTK pseudokinases is required for their signaling activity in cells, and that they can be inhibited with ATP-competitive inhibitors. Addressing these questions will shed important new light on the mechanism of signaling by RTK pseudokinases, which have all been associated with human cancer. If our hypothesis is correct, the findings will lay the foundation for developing a new set of TKIs against long-neglected targets that would have very significant clinical impact. If the hypothesis is incorrect, our studies will provide valuable structural and biochemical frameworks for exploring (and inhibiting) the unique signaling mechanisms of these receptors.